A characteristic feature of segmented RNA viruses is that they can undergo reassortment during host cell co-infection. This process results in reassortant viral progeny with genome segments derived from more than one parental strain. In some cases, the new segment combination confers a selective advantage to the virus, allowing it to emerge in the population. However, some viral strains are incapable of reassorting with each other even during experimental co-infection, and other strains show strong biases towards reassorting only a few genome segments. These observations suggest that reassortment requires genetic compatibility among the viral RNAs and/or their encoded proteins. Nucleotide and/or amino acid differences between parental strains represent reassortment restriction determinants if they prevent either the generation of reassortants during co-infection or the emergence of reassortants in the viral population. Here, a genetic approach will be employed to identify reassortment restriction determinants for rotaviruses, 11-segmented, double-stranded RNA viruses that cause life-threatening diarrhea in young children. In Aim 1, a fully plasmid-based reverse genetics system will be used to determine the extent of genetic compatibility among heterologous rotavirus genome segments and to map the locations of nucleotides and/or amino acids that dictate segment incompatibility. In Aim 2, an innovative in vitro genome segment packaging assay will be developed and used to define whether nucleotide differences directly inhibit the co-packaging of heterologous strain RNA molecules. This work is significant because it will elucidate novel functional connections among rotavirus genes/proteins during viral replication and enhance an understanding of the factors promoting or tempering rotavirus evolution. Residues shown to impact reassortment could be manipulated to engineer genetically-stable, live-attenuated vaccine candidates that cannot exchange genome segments with wildtype strains. This work may also illuminate features of rotavirus replication and evolution that are shared with other segmented RNA viruses, thereby informing the development of broad-spectrum therapeutic interventions. Thus, this work represents the first step in a continuum of research aimed at developing new strategies to prevent and treat viral disease.